Novel therapies are need for the treatment of triple negative breast cancer (TNBC). In response to this clinical need, this proposal will test the efficacy of a novel combinatorial strategy which targets differentiated TNBC cells and TNBC cancer initiating cells (CICs). The latter identified as ALDHbright cells have to be eradicated in order to cure a malignant disease, since according to cancer stem cell theory these cells are responsible for disease recurrence and metastases. The tumor antigen selected as a target is the glucose-regulated protein of 94 kDa (Grp94). Grp94 regulates the activation of signaling pathways associated with cell proliferation, survival and migration. To target Grp94, we will take advantage of the unique specificity of the human mAb W9 we have recently characterized. mAb W9 recognizes an extracellular Grp94 epitope selectively expressed on cancer cells, including differentiated TNBC cells and TNBC CICs, but with a restricted distribution in normal tissues. mAb W9 markedly inhibits TNBC and TNBC CICs in vitro proliferation. To enhance its anti-proliferative activity we have combined mAb W9 with LDE225, a novel inhibitor of the Sonic Hedgehog (SHH) pathway which is aberrantly activated in TNBC and especially in TNBC CICs. mAb W9 enhances the ability of LDE225 in inhibiting TNBC cell in vitro growth, and in eliminating TNBC CICs by inhibiting signaling pathways involved in cell proliferation and survival of TNBC cells and TNBC CICs. The in vivo and potential clinical relevance of the in vitro results will be assessed by the following specific aims: i) mAb W9 and LDE225 combination is more effective than the individual agents in eradicating TNBC tumors established in Severe Combined Immunodeficiency (SCID) mice orthotopically grafted with the TNBC cell line MDA-MB-231-Luc-D3H1; ii) the results obtained with the TNBC cell line MDA-MB-231-Luc-D3H1 have clinical significance, as they are reproduced in SCID mice orthotopically grafted with TNBC tumors surgically removed from patients. The results generated by the outlined experiments will contribute to determine whether mAb W9 should be moved to a clinical setting. Its translation will be facilitated by the fact that the mAb W9 is a fully human antibody and does not require any major manipulation for use in patients. Furthermore, the clinical implementation of this novel combinatorial strategy will be facilitated b Dr. Isakoff's access to his patients with TNBC and his skills in conducting clinical trials.